The proposed Atlantic Coast (AC) STI CRC is a highly integrated, multidisciplined alliance of experienced investigators who are committed to advancing basic and translational science in the area of Neisseria gonorrhoeae (Ng), Chlamydia trachomatis (Ct) and Ng/Ct infections that will further our understanding of the pathogenesis of these sexually transmitted infections and lead to the development of new interventions. To meet these goals, we will address the following four programmatic specific aims. Aim 1 is to define the immunobiology of Ng and Ng/Ct co-infection with respect to mechanisms of Ng-mediated immunosuppression and the mechanisms by which Ng and Ct evade or interact with effectors of the innate defense. Two novel Ng immunosuppressive pathways that interfere with dendritic cell (DC) function will be characterized. The hypothesis that these Ng pathways affect host responses to Chlamydia will also be tested. A vaginal proteome analysis will be performed using mouse models of single and dual infection to identify novel effectors that may limit or enhance infection and to assess the influence of reproductive hormones on the expression of host factors. We will also investigate the role of perforin-2, a membrane-bound pore-forming effector, in the intracellular biology of Ng, Ct and Ng/Chlamydia at the cellular level and during experimental murine infection. Aim 2 is to develop tissue culture systems, improved animal models, and immunological systems for studying Ng/Ct confection and to accelerate product development. Tissue culture systems designed to promote Ng invasion through two distinct pathways and sophisticated microscopy will be utilized to examine the intracellular biology of Ng/Ct coinfected cells. Improved mouse models will be established using human transferrin-supplemented mice that are transgenic for the human carcinoembryonic antigen-related cellular adherence molecule (hCEACAM-1); these models will be used to identify potential biomarkers that can discriminate single and dual infections. To facilitate studies on the immunobiology of co-infection, in vitro, ex vivo and in vivo assays will be developed to examine the effect of Ng on DC-directed anti-Cm responses. Aim 3 is to understand the genetic basis of the spread of antibiotic resistant gonorrhea. The effect of ceftriaxone resistance determinants on microbial fitness in vitro and in vivo will be tested and compensatory mutations that restore fitness will be identified. Aim 4 is to develop a gonorrhea vaccine and novel therapies effective against both gonorrhea and Chlamydia infections. A promising Ng Tf receptor vaccine will be developed and the effect of a pre-existing Chlamydia infection on the efficacy of this vaccine will be assessed. Iron-chelators will be tested as a nutritional therapy and a prototype histone deacetylase inhibitor will be tested as a potentially effective epigenetic therapy that induces the expression f innate effectors and suppresses pathogen-induced inflammation.